The present invention relates, first, to methods for the modulation of acid sphingomyelinase (ASM)-related processes, including apoptosis. Such apoptosis can include, but is not limited to, environmental stress-induced apoptosis such as, for example, ionizing radiation and/or chemotherapeutic agent-induced apoptosis. Apoptosis can be characterized by a cellular morphology comprising cellular condensation, nuclear condensation or zeiosis. The present invention further relates to methods for the identification of compounds which modulate (i.e., either increase or decrease) sensitivity to ASM-related processes, including apoptosis.
The sphingomyelin pathway is a ubiquitous, evolutionarily conserved signaling system initiated by hydrolysis of sphingomyelin to generate the second messenger ceramide. Two forms of sphingomyelinase, distinguishable by the pH optima, are capable of initiating signaling. Acid sphingomyelinase (pH optimum 4.5-5.0) was originally identified as a lysosomal hydrolase required for turnover of cellular membranes (for review see Kolesnick, R. N., 1991, Prog. Lipid Res. 30 1-38). However, Kronke and co-workers proposed that this enzyme was also targeted to the plasma membrane and signaled in response to activation of the 55 kD TNF receptor (Wiegmann, K. et al., 1994, Cell 78:1005-1015). Activation of acid sphingomyelinase has also now been associated with signaling via Fas, CD28 and the interleukin (IL)-1 receptor (Cifone, et al., 1993., J. Exp. Med. 177:1547-1552; Boucher, L. -M. et al., 1995, J. Exp. Med. 181:2059-2068.; Liu. P. and Anderson, R. G. W., et al., 1995, J. Biol. Chem. 270:27179-27185. Human acid sphingomyelinase is the product of a single gene, although alternative processing of the primary transcript allows for the generation of multiple forms (Schuchman, E. H., et al., 1991, J. Biol. Chem. 266:8531-8539; Schuchman, E. H., et al., 1992, Genomics 12:197-205. Inherited mutations of the human acid sphingomyelinase gene lead to enzyme deficiency and the genetic disorder known as Niemann Pick disease (NPD; Brady et al., R. O., et al., 1966, Proc. Natl. Acad. Sci. USA 55, 366-369.; Schneider, P. B. and Kennedy, E. P., 1967, J. Lipid Res. 8:202-209.
Neutral sphingomyelinase (pH optimum 7.4) was originally defined as a Mg2+-dependent enzyme localized to the outer leaflet of the plasma membrane (Rao, B. G. and Spence, M. W., 1976, J. Lipid Res. 17:506-515.; Yedger, S. and Gatt, S., 1976. Biochemistry 15:2570-2573. However, a Mg2+-independent isoform of neutral sphingomyelinase which localizes to the cytoplasm has recently been identified (Okazaki, T. et al., 1989, J. Biol. Chem. 264:19076-19080.; Okazaki, T. et al., 1994, J. Biol. Chem. 269:4070-4077.). The neutral sphingomyelinase has not yet been characterized at the molecular level.
Neutral sphingomyelinase activation has been demonstrated in response to cellular stimulation with TNFa (Wiegmann, K. et al., 1994, Cell 78:1005-1015) anti-Fas antibody (Tepper, C. G., et al., 1995, Proc. Natl. Acad. Sci. USA 92:8443-8447.; Cifone, M. G. et al., 1995, EMBO J. 14:5859-5868.), and vitamin D (Okazaki, T. et al., 1989, J. Biol. Chem. 264:19076-19080; Okazaki, T. et al., 1994, J. Biol. Chem. 269:4070-4077.). It has also been suggested that neutral sphingomyelinase signals in response to IL-1b (Mathias, S. et al., 1993, Science 259:519-522) and ionizing radiation (Halmovitz-Friedman, A. et al., 1994, J. Exp. Med. 180:525-535.).
Although ceramide has been implicated as the second messenger for a variety of stress stimuli including TNFa, Fas ligand, ionizing radiation, heat shock, ultraviolet light and oxidative stress (Obeid, L. M. et al., 1993, Science 259:1769-1771.; Cifone, M. G. et al., 1993, J. Exp. Med. 177:1547-1552.; Jarvis, W. D., et al., 1994, Proc. Natl. Acad. Sci. USA 91:73-77; Fuks, Z., et al., 1994, Cancer Res. 54:2582-2590; Halmovitz-Friedman, A., et al., 1994, J. Exp. Med. 180:525-535.; Gulbins, E. et al., 1995, Immunity 2:341-351.; Verheij, M. et al., 1996, Nature 380:75-79; Jarvis, W. D. et al., 1995, Grant, S., Clin. Cancer Res. 2:1-6). Evidence for such speculation has been circumstantial (Verheij, M. et al., 1996, Nature 380:75-79; Hannun, Y. A. and Obeid, L. M, 1995, Trends Biochem. Sci. 20:73-77). Definitive proof, therefore, that ceramide generation is a primary mediator of the apoptotic response is lacking.
The present invention relates, first, to methods for the modulation of acid sphingomyelinase (ASM)-related processes, including apoptosis. Such apoptosis can include, but is not limited to, environmental stress-induced apoptosis such as, for example, ionizing radiation and/or chemotherapeutic agent-induced apoptosis. Apoptosis can be characterized by a cellular morphology comprising cellular condensation, nuclear condensation or zeiosis.
The present invention is based, in part, on the surprising discovery, described in the Example presented in Section 6, below, that acid sphingomyelinase activity is required for activation of stress-induced apoptotic cellular pathways. Specifically, the data presented in these Examples shows that ASM-deficient cell lines and ASM-deficient animals are resistant to radiation-induced apoptosis. Thus, the data described herein define, for the first time, an obligatory role for ceramide generation in signalling of stress-induced apoptosis.
The present invention further relates to methods for the identification of compounds which modulate ASM-related processes, including apoptosis. xe2x80x9cModulationxe2x80x9d as used herein, can refer, first, to an increase in the sensitivity of cells, especially neoplastic cells, to ASM-related processes, including apoptosis. Alternatively, xe2x80x9cmodulationxe2x80x9d can refer to a decrease in the sensitivity of cells to ASM-related processes such as apoptosis; e.g., can refer to an increase in the cells"" resistance to apoptosis.
Methods for the identification of compounds which increase a cell""s sensitivity to ASM-related processes such as apoptosis can be performed to identify targets and compounds which mimic ASM or act downstream of ASM in apoptotic pathways. Among the compounds and targets identified via such identification methods are agents which can be utilized to increase a neoplastic cell""s sensitivity to apoptosis, thereby improving the clinical effects of anti-cell proliferative therapy, e.g. radiation and/or chemotherapeutic therapies.
Such methods can include, for example, a method comprising, first contacting an acid sphingomyelinase-deficient cell with a test compound, exposing the cell to a stress stimulus for a time sufficient to induce apoptosis in a cell exhibiting normal acid sphingomyelinase activity. Second, an acid sphingomyelinase-deficient cell is exposed, in the absence of the test compound, to the stress stimulus for a time sufficient to induce apoptosis in a cell exhibiting normal acid sphingomyelinase activity. The exposed cells are monitored for the presence of an apoptotic morphology, such that if the cell exposed to the test compound exhibits a more severe apoptotic morphology, the test compound represents a compound which increases a cell""s sensitivity to acid sphingomyelinase-related apoptosis.
Alternatively, such methods for identifying a compound which increases a cell""s sensitivity to acid sphingomyelinase-related apoptosis can also comprise, first, contacting an acid sphingomyelinase-deficient cell with a test compound, and exposing the cell to a stress stimulus. Next, an acid sphingomyelinase-deficient cell is exposed, in the absence of the test compound, to the stress stimulus. The levels of sphingomyelin and ceramide present in the exposed cells are compared, such that if the level of sphingomyelin in the cell exposed in presence of test compound is less than that of the cell exposed in the absence of the test compound, or the level of ceramide in the cell exposed in the presence of test compound is greater than that of the cell exposed in the absence of test compound, the test compound represents a compound which increases a cell""s sensitivity to acid sphingomyelinase-related apoptosis.
The cells utilized in the above-described methods for identifying compounds which increase a cell""s sensitivity to ASM-related apoptosis can be part of a genetically engineered nonhuman animal deficient for the acid sphingomyelinase gene, such that the animal is exposed to the stress stimulus, either in the presence or absence of test compound.
Additionally, methods for the identification of compounds which decrease a cell""s sensitivity to ASM-related processes such as apoptosis can be performed. Such screens can identify additional targets in the apoptotic pathway which, like ASM, are necessary for stress-induced apoptosis to occur. Further, such screens can identify compounds useful for minimizing the effects of stress-induced apoptosis, for example, apoptosis induced by radiation.
Such methods for identifying a compound which decreases a cell""s sensitivity to acid sphingomyelinase-related apoptosis can include, for example, a method comprising, first, contacting a cell exhibiting acid sphingomyelinase activity with a test compound and exposing the cell to an apoptosis-inducing stress stimulus. Next, a cell which exhibits acid sphingomyelinase activity is exposed, in the absence of test compound, to the stress stimulus. The exposed cells are monitored for the presence of an apoptotic morphology, such that if the cell exposed in the presence of the test compound exhibits a less severe apoptotic morphology, than the cell exposed in the absence of the test compound, the test compound represents a compound which decreases a cell""s sensitivity to acid sphingomyelinase-related apoptosis.
Such methods for identifying a compound which decreases a cell""s sensitivity to acid sphingomyelinase-related apoptosis, can also include, for example, a method comprising, first, contacting a cell exhibiting acid sphingomyelinase activity with a test compound, and exposing the cell to a stress stimulus. Next, a cell exhibiting acid sphingomyelinase activity is exposed, in the absence of test compound, to the stress stimulus. The levels of sphingomyelin and ceramide present in the exposed cells are compared such that if the level of sphingomyelin in the cell exposed in the presence of test compound is greater than that of the cell exposed in the absence of test compound, or the level of ceramide in the cell exposed in the presence of test compound, is less than that of the cell exposed in the absence of test compound, the test compound represents a compound which decreases a cell""s sensitivity to acid sphingomyelinase-related apoptosis.
In the above-described methods for identifying compounds which decrease a cell""s sensitivity to ASM-related apoptosis, the cells utilized can be transgenic cells comprising cells deficient in endogenous acid sphingomyelinase gene activity and containing a functional human acid sphingomyelinase transgene capable of expressing functional human acid sphingomyelinase. Further, such cells can be part of genetically engineered nonhuman animal deficient in endogenous acid sphingomyelinase gene activity and containing integrated in its cells a functional human acid sphingomyelinase transgene capable of expressing functional human acid sphingomyelinase.
In the above-described methods for identifying compounds which decrease a cell""s sensitivity to ASM-related apoptosis, the cells utilized can also be genetically engineered cells which exhibit a greater level of acid sphingomyelinase activity than non-genetically engineered cell of the same type.